Acetyl coenzyme A (acetyl-CoA) is a key intermediate in the synthesis of essential biological compounds, including polyketides, fatty acids, isoprenoids, phenolics, alkaloids, vitamins, and amino acids. Among the metabolites derived from acetyl-CoA are primary and secondary metabolites, including compounds of industrial utility. Isoprenoids, for example, are used in pharmaceutical products and as biofuels, food additives, and other specialty chemicals. An isoprenoid product is typically composed of repeating five carbon isopentenyl diphosphate (IPP) units, although irregular isoprenoids and polyterpenes have been reported. In nature, isoprenoids are synthesized by consecutive condensations of their precursor IPP and its isomer dimethylallyl pyrophosphate (DMAPP). Two pathways for these precursors are known. Prokaryotes, with some exceptions, typically employ the deoxyxylulose-5-phosphate (DXP) pathway to convert pyruvate and glyceraldehyde 3-phosphate (G3P) to IPP and DMAPP. Eukaryotes, with the exception of plants, generally use the mevalonate-dependent (MEV) pathway to convert acetyl-CoA to IPP, which is subsequently isomerized to DMAPP.
The unicellular fungus Saccharomyces cerevisiae and its close relatives use two endogenous pathways to generate acetyl-CoA. One pathway takes place in the mitochondrial matrix, where the PDH complex catalyzes the oxidative decarboxylation of pyruvate, generated from glucose via glycolysis, to acetyl CoA. The PDH complex consists of 60 polypeptide chains—24 chains of the lipoamide reductase-transacetylase, 12 chains of dihydrolipyl dehydrogenase, and 24 chains of pyruvate decarboxylase. This massive complex converts pyruvate to acetyl-CoA, generating NADH as a byproduct. The resulting acetyl-CoA can then be completely oxidized to CO2 and H2O via the citric acid cycle for energy generation, or be used for biosynthetic reactions that are performed in the mitochondria.
The acetyl-CoA generated in the mitochondria is unable to cross the mitochondrial membrane into the cytosol. Thus, to generate cytosolic acetyl-CoA, which is needed for the biosynthesis of important primary and secondary metabolites, S. cerevisiae uses an independent mechanism located in the cytosol known as the “PDH-bypass.” This multi-step pathway catalyzes: (1) the decarboxylation of pyruvate into acetaldehyde by pyruvate decarboxylase (PDC, EC 4.1.1.1); (2) the conversion of acetaldehyde into acetate by acetaldehyde dehydrogenase (ACDH, EC 1.2.1.5 and EC 1.2.1.4), which reduced one NADP+ to one NADPH; and (3) the synthesis of acetyl-CoA from acetate and CoA by acetyl-CoA synthetase (ACS, EC 6.2.1.1), which hydrolyzes 1 ATP to 1 AMP, the energetic equivalent of hydrolyzing 2 ATP to 2 ADP.
Since nature provides only low yield sources for the extraction of many acetyl-CoA derived biomolecules, fermentative production using genetically modified microorganisms has become a promising alternative for their production. However, utilization of the native acetyl-CoA pathway for production of the acetyl-CoA intermediate has certain limitations. For example, isoprenoid production via the native MEV pathway requires three acetyl-CoA molecules and the oxidation of two NADPH for each molecule of mevalonate generated, as shown in FIG. 1. While the PDH-bypass generates one NADPH per acetyl-CoA produced, two ATP equivalents are expended in the process. Thus, while the generation of NADPH is beneficial with regard to the cofactor requirements of the native MEV pathway, the expenditure of six ATP equivalents per mevalonate generated results in an energetically inefficient reaction, as more carbon source must be diverted to ATP synthesis, e.g., via the TCA cycle and oxidative phosphorylation, at the expense of product yield.
Thus, one of the challenges in designing a production host that efficiently produces acetyl-CoA derived compounds is to optimize acetyl-CoA production such that the ATP requirements are minimized, while also meeting the co-factor and requirements of the biosynthetic pathway. The compositions and methods provided herein address this need and provide related advantages as well.